The present invention relates to a method for the selective removal of contaminants, including aflatoxins, from azadirachtin-containing materials. More particularly, this invention relates to a method of reducing the aflatoxin content of azadirachtin-containing materials through the selective binding of aflatoxin by charcoal. Charcoal is defined as a black form of carbon produced by partially burning wood, coal, lignin, bone or other organic matter in a kiln from which air is excluded. Some of the synonyms for charcoal include carbon, activated carbon, and activated charcoal. Charcoal may be prewashed with acid or base with or without subsequent neutralization and may be in powder, granular or pelletized forms.
Extracts of the neem (Azadirachta indica) and the chinaberry (Melia azedarach) trees have long been known to have insecticidal activity (Natural Pesticides from the Neem Tree, Proc. 1st Int'l Neem Conf., Rottach-Egern, 1980 (H. Schmutterer, et al. eds. 1982); Natural Pesticides from the Neem Tree and Other Tropical Plants, Proc. 2nd Int'l Neem Conf., Rauisch holzhausen, 1983 (H. Schmutterer and K.R.S. Asher eds. 1984); Natural Pesticides from the Neem Tree and Other Tropical Plants, Proc. 3rd Int'l Neem Conf., Nairobi, 1986 (H. Schmutterer and K.R.S. Asher eds. 1987)). These extracts have generated intense academic research interest including the isolation and identification of at least sixty different chemical entities from various parts of the neem tree (P. Jones, et al., "The Chemistry of the Neem Tree," in Phytochemical Pesticides, Vol. 1 (M. Jacobson ed., CRC Press, 1988).
The active ingredient of the neem and chinaberry extracts, azadirachtin, is a limonoid of the tetranortriterpenoid type. This compound has been shown to be a potent insect growth regulator and feeding deterrent (Yamasaki, R. B., et al. (1987) J. Agric. Food Chem. 35:467-471). Several synthetic analogs of azadirachtin have been prepared (Yamasaki, R. B., et al. (1987) suora); Ley, S. V., et al. (1989) Tetrahedron 45:5175) and organic synthesis of the molecule has been accomplished (Ley, S. V., et al. (1987) Tetrahedron Lett. 28:221; Brasca, M. G., et al. (1988) Tetrahedron Lett. 29:1853; Ley, S. V., et al. (1989) Tetrahedron 45:2143; Nishikimi, Y., et al. (1989) J. Org. Chem 54:3354). Due to the complexity of the azadirachtin molecule, however, the economic synthesis and commercialization of a synthetic product is highly unlikely and therefore any salable product will require extraction from an azadirachtin-containing plant source.
Recently, two companies have received approvals for the sale of azadirachtin-containing neem seed extracts in the United States. The importance and attractiveness of these and future azadirachtin-containing extracts is due to their natural source, broad spectrum of insecticidal activity (Rembold, H., et al. (1981) Naturforsch C: Biosci. 36:466-469), lack of insect resistance, nonmutagenicity (Jacobson, M., Natural Pesticides from the Neem Tree, Proc. 1st Int'l Neem Conf., supra, pp. 33-42), and low mammalian toxicity (Nakanishi, K. (1975) Rec. Adv. Phytochem. 9:283-298).
Although the extraction of azadirachtin from plants and seeds has both economic and ecological advantages over organic synthesis, these extracts have the disadvantage of being easily susceptible to contamination with aflatoxins. These mycotoxins, produced by the fungi Aspergillus flavus and Aspergillus parasiticus, are known for their severe acute toxicity and potent carcinogenicity (Sargeant, et al. (1961) Nature 192:1096). Aflatoxin contamination is common in many agricultural commodities (e.g., corn, groundnuts, milk, dried chili peppers, cottonseed meal, coconut oil), including neem products, due to the growth of the fungi before and after harvesting and processing. During the extraction of azadirachtin from plants and seeds, aflatoxins are extracted and concentrated along with the desired compounds, specifically azadirachtin. Without removal, these aflatoxins could present serious health threats to the handlers and users of azadirachtin-containing extracts. Consequently, the presence of high levels of aflatoxin in commercial plant extracts render the material unacceptable to the producer and user because of health concerns.
Various methods of reducing the aflatoxin content of edible oils using physical and chemical techniques have been proposed. In the physical decontamination methods, the decomposition of aflatoxin is generally accomplished by the use of dry heat (Mann, G. E., et al. (1967) J. Agr. Food Chem. 15:1090; Lee, L. S., et al. (1969) J. Agr. Food Chem. 17:451) or moist heat (Coomes, T. J., et al. (1966) Nature 209:406). Chemical decontamination methods involve the decomposition of aflatoxin using ammonia (Gardner, H. K., et al. (1971) J. Am. Oil Chem. Soc'y 48:70; Masri, M. S., et al., U.S. Pat. No. 3,429,709), sodium hypochlorite (Fischbach, H., et al. (1965) J. Assoc. Off. Agr. Chemists 48:28), chlorine gas (Goldblatt, L. A. (1965) Abst. Meeting Am. Chem. Soc'y, 150th, p. 5a), hydrogen peroxide (Sreenivasamurthy, V., et al. (1967) J. Ass. Off. Anal. Chem. 50:350), methyl amine (Godfrey, E., et al., U.S. Pat. No. 3,585,041), liquid dimethyl ether and water (Yano, N., et al., U.S. Pat. No. 4,055,674), alkaline and acid solutions (Watanabe, H., et al., U.S. Pat. No. 4,280,962), mixtures of acetone, hexane and water (Goldblatt, L. A., et al., U.S. Pat. No. 3,515,736), and mixtures of an alkali or alkaline earth metal and an organic amine (Brandt, J., et al., U.S. Pat. No. 3,890,452). All of these methods degrade aflatoxin quite efficiently, but they have the inherent disadvantage of also degrading azadirachtin. These methods are therefore commercially impractical for the purification of azadirachtin-containing materials.
Methods of detoxication of aflatoxin-contaminated edibles using microbiological and adsorptive techniques have also been proposed. It has been reported, for example, that the nonpathogenic bacterium, Flavobacterium aurantiacum, somehow inactivates or removes aflatoxins from contaminated agricultural products (Ceigler, A. (1966) Appl. Microbiol. 14:934; Ceigler, A., et al., U.S. Pat. No. 3,428,458). Adsorptive methods for aflatoxin removal include the use of microbial mycelium (Masimango, N., et al. (1978) Eur. J. Appl. Microbiol. Biotechnology 6:101; Masimango, N., et al. (1979) Ann. Nutr. Aliment. 33:149), swollen clays (Masimango, N., et al. (1979) Ann. Nutr. Aliment. 33:137), and coconut wastes (Kumari, C. K., et al. (1987) Res. & Indus. 32:85). It is significant to note that these prior art methods all involve prolonged, costly and/or inefficient procedures and, like the chemical and physical detoxication methods, are commercially impractical for the treatment of extracts containing azadirachtin.
German Offen. No. 2,627,613 discloses the purification of a protein extract from oil seeds by treating it with activated carbon. The protein extract is reportedly deodorized and decontaminated by passing the extract through alkaline-pretreated activated carbon, eluting with alkaline solution, and treating the effluent with acid solution. Like the chemical treatments previously mentioned, this method is impractical for the purification of azadirachtin-containing compositions since azadirachtin, like aflatoxin, is degraded in alkaline solutions.
Administration of activated charcoal reportedly reduces the effects of aflatoxicosis in chickens. Dalvi, R., et al. (1984) Avian Dis. 28(1):61-69; Dalvi, R., et al. (1984) Poult. Sci. 63(3):485-491; Ademoyero, A., et al. (1983) Toxicol. Lett. 16(1-2):153-157. Similarly, goats afflicted with acute aflatoxicosis reportedly respond to treatment with activated charcoal or dual combinations of oxytetracycline or stanozolol and activated charcoal. Hatch, R. C., et al. (1982) Am. J. Vet. Res. 43(4):644-648. The removal of aflatoxin from aqueous solutions of the sugar xylitol by the use of charcoal has been demonstrated. Frank, P., (1972) Deut. Apoth. -2tg. 112(22):838. Although these references teach the reversal of aflatoxicosis and the absorption of aflatoxin from aqueous sugar solutions, none teach the selective removal of aflatoxin from organic or mixed organic/aqueous solutions in the presence of azadirachtin.
A need therefore exists for a neem extract purification process which effectively and selectively removes aflatoxin, without concomitantly affecting azadirachtin content, and is practical and economical in operation.